This is a revised competing renewal of an R01 grant to characterize the function of the murine gamma herpes virus 68 (MHV68;also known as 3HV68) M2 antigen. Gamma-herpesviruses are associated with the development of lymphoproliferative disorders and lymphomas, particularly in immunosuppressed individuals. Importantly, a detailed understanding of how chronic virus infection is established and maintained may identify possible targets for interfering with viral persistence in the host. Unfortunately, the human gammaherpesviruses (EBV and KSHV) have very narrow host tropism, thus limiting detailed studies of host-pathogen interactions. MHV68 infection of mice provides a tractable small animal model system for characterizing the role of specific genes in viral pathogenesis and maintenance of chronic infection. We have previously shown that the MHV68 M2 latency associated antigen plays a critical role in both the establishment of latency and reactivation of MHV68 from infected B cells. Furthermore, our recent studies have shown that M2 induces an IL-10 dependent proliferation and differentiation of primary murine B cells - reminiscent of EBV-driven differentiation of primary human B cells. M2 regulation of cellular IL-10 expression is particularly intriguing given that EBV encodes an IL-10 homolog whose function during virus infection has remained enigmatic. In addition, M2 can drive terminal differentiation of latently infected B cells to plasma cells, with concomitant reactivation of MHV68. Notably, plasma cell differentiation has also been linked to EBV and KSHV reactivation - indicating that virus regulation of plasma cell differentiation is likely a conserved strategy among gammaherpesviruses. Further characterization of MHV68 M2 antigen function are expected to reveal cellular pathways that are manipulated by gamma-herpesviruses to facilitate persistence and reactivation from B cells, and may ultimately identify targets for the development of anti-viral drugs. Our studies on the role that the M2 antigen plays in chronic MHV68 infection will focus on the following aims: Aim 1 - Regulation of M2 gene transcription: 1.a. Identification of cell types expressing M2 in vivo;1.b. Analysis of viral gene expression in MHV68 latently infected cells expressing M2;1.c. Map and characterize cis-elements regulating M2 gene expression during latency and reactivation. Aim 2 - Further characterization of M2 mutants: 2.a. Generation and characterization of single tyrosine mutants;2.b. Impact of tyrosine and PxxP mutations on virus latency and reactivation;2.c. Assess requirement for M2 post-establishment of latency;2.d. Generation and characterization of chimeric LMP2A-M2 proteins. Aim 3 - Identification of M2 interacting proteins: 3.a. Role of M2 phosphorylation and interaction with SH2 domain containing proteins;3.b. Interaction of SH3 and WW domain containing proteins with functionally important proline rich domains in M2. Aim 4 - Modulation of cellular signaling by M2: 4.a. M2 induction of IL-10 expression;4.b. M2 regulation of plasma cell differentiation;4.c. M2 induced changes in cellular gene expression. PUBLIC HEALTH RELEVANCE Murine gammaherpesvirus 68 (MHV68) infection of mice provides a tractable small animal model for characterizing chronic gammaherpesvirus infections - which in some settings (e.g., immunocompromised individuals) are associated with the development of lymphoproliferative disorders and lymphomas, as well as several other cancers. The importance of this model is underscored by the narrow host tropism of the human gammaherpesviruses, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, and thus the absence of appropriate animal models for these viruses. During the previous funding period we have discovered that expression of the MHV68 latency-associated M2 antigen in B cells drives cellular proliferation, IL-10 expression, antibody class switch recombination and plasma cell differentiation - the latter being linked to virus reactivation. Although the M2 antigen is unique to MHV68, the functions of M2 have close parallels in the human gammaherpesviruses - indicating that further characterization of the M2 antigen will provide important new insights into cellular pathways that are targeted by gammaherpesviruses and may ultimately identify targets for the development of antiviral therapies effective against chronic gammaherpesvirus infections.